Effects of HMGB1 on PMN apoptosis during LPS-induced acute lung injury.
OBJECTIVE
To observe the effects of intravenous injection of HMGB1 inhibitor sodium butyrate on changes in apoptosis of PMN during LPS-induced acute lung injury in rats and HMGB1 in vitro on human circulating PMN apoptosis, in order to clarify the role of HMGB1 in the pathogenesis of acute lung injury.


METHODS
(1) LPS-induced acute lung injury rat model was developed by LPS infusion. At different time-points after LPS challenge in the presence or absence of sodium butyrate (SB), the rat tissue sample, peripheral blood PMNs and BALF were collected. RT-PCR was applied to examining rat lung tissue HMGB1 mRNA expression level, and Western blotting analysis was adopted to determine expression of rat lung tissue HMGB1 protein. PMN apoptotic changes were determined by flow cytometric (FCM) analysis, Giemsa staining and TdT-mediated dUTP nick end labeling (TUNEL) method. (2) Separated and purified human circulating PMN were coincubated for 24 h with different doses of HMGB1 (0, 10, 100, 1000 ng/ml, respectively) at 37 degrees C in 5% CO(2). PMN apoptosis rate was determined by flow cytometric (FCM) analysis and by TdT-mediated dUTP nick end labeling (TUNEL) method.


RESULTS
(1) The percentage of apoptosis of PMN in rat model of LPS-induced ALI was gradually decreased as compared with that of normal control. The PMN apoptosis-initiation time and non-survival time in rat BALF prolonged significantly as compared with that of normal control. The injured rat lung tissue HMGB1 mRNA and protein expression was upregulated 6-24 h after LPS exposure; SB intervention significantly ameliorated the upregulation. In addition, the morphologic examination indicated that the edema severity and pathological changes of lung tissues were excessively aggravated in rats after LPS administration. By comparison, SB treatment diminished the severity of lung damage. Combined with lung HMGB1 expression level, the above changes indicate that the pathological changes of lung tissue were related to the injured lung HMGB1 expression, as well as apoptotic changes in PMN. (2) After coincubation of HMGB1 with human circulating PMNs, TUNEL and flow cytometry were performed. The study revealed that PMN apoptosis ratios was (40.53+/-4.12) % in control group (PMNs+RPMI 1640 medium), (19.05+/-2.44) % in LPS group (PMNs+RPMI 1640 medium+10 microg/ml LPS), (40.52+/-2.73) % in HMGB1-1 group (PMNs+RPMI 1640 medium+10 ng/ml HMGB1), (34.89+/-1.15) % in HMGB1-2 group (PMNs+RPMI 1640 medium+100 ng/ml HMGB1), and (18.77+/-3.02) % in HMGB1-3 group (PMNs+RPMI 1640 medium+1 000 ng/ml HMGB1). There was statistical significance. Meanwhile, PMN TUNEL positive rate was (31.42+/-4.40) %, (31.39+/-3.80) %, (25.62+/-2.46) %, and (17.98+/-3.20) % in control group, HMGB1-1 group, HMGB1-2 group and HMGB1-3 group, respectively. The inhibitory effect was HMGB1 dose-depended as compared with that of control group.


CONCLUSIONS
After LPS challenge, high expression of rats' lung HMGB1 mRNA occurs at a later phase, but keeps for a long time. Sodium butyrate (SB) treatment attenuated LPS-induced PMN apoptosis delay and inhibition, and down-regulated HMGB1 mRNA expression of injured lung. HMGB1 in vitro inhibited human circulating PMN apoptosis markedly, and the inhibitory effect was HMGB1 dose-depended. The results demonstrated that HMGB1 may play an important role as a modulator in apoptotic changes in PMN during LPS-induced ALI. It concludes that HMGB1 may contribute to the development of PMN apoptotic changes during LPS-induced acute lung injury.
Feng
Yingkai
Y
Institute of Pathology, Southwest Hospital Affiliated to Third Military Medical University, Chongqing 400038, China.
Yang
Qinghua
Q
Xu
Jiancheng
J
Qian
Guisheng
G
Liu
Yousheng
Y
D055371:Acute Lung Injury / Q000139:chemically induced / Q000473:pathology*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001399:Azure Stains; D001992:Bronchoalveolar Lavage Fluid / Q000166:cytology; D002470:Cell Survival / Q000187:drug effects; D005434:Flow Cytometry; D006098:Granulocytes / Q000187:drug effects* / Q000473:pathology*; D024243:HMGB1 Protein / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D020287:In Situ Nick-End Labeling; D007275:Injections, Intravenous; D008070:Lipopolysaccharides; D008168:Lung / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D008297:Male; D009929:Organ Size / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D013997:Time Factors
D001399:Azure Stains; D024243:HMGB1 Protein; D008070:Lipopolysaccharides; D012333:RNA, Messenger